Luminex magpix detection system

The substantia nigra was homogenized with the protein extraction buffer containing 100 mM Tris-HCl (pH 7.4), 750 mM NaCl (sodium chloride), 10 mM EDTA (ethylenediaminetetraacetic acid), 5 mM EGTA (ethylene glycol tetraacetic acid), and protease inhibitors (Roche, Basel, Switzerland) [5 (link), 45 (link)]. The samples were centrifuged at 1000g for 10 min at 4°C. The supernatant was collected and centrifuged again at 20,000g for 40 min at 4°C to remove remaining debris. ELISA was performed using a Milliplex MAP Rat cytokine/chemokine magnetic bead panel kit according to the provider's protocol (RECYTMAG_65K; Millipore, Temecula, CA, USA), and reading was made by using the LUMINEX MAGPIX detection system with xPONET software (Millipore Corporation, Billerica, MA, USA). The sensitivity ranges were 2.4 to 10,000 pg/mL for TNF-α and IL-1β, 73.2 to 300,000 pg/mL for IL-6, and 7.3 to 30,000 pg/mL to IL-4 and IL-10.

Homogenization of brain tissues (striatum and SN) was made using protein extraction buffer containing 100 mM Tris HCl (pH 7.4), 750 mM NaCl (sodium chloride), 10 mM EDTA (ethylenediaminetetraacetic acid), 5 mM EGTA (ethylene glycoltetraacetic acid) and a cocktail of protease inhibitors (Roche, Basel, Switzerland) [57 (link)]. The samples were centrifuged at 1,000 g for 10 minutes at 4°C and then supernatant was centrifuged again at 20,000 g for 40 minutes at 4°C to remove the remaining debris. For detecting the levels of inflammatory cytokines, indirect ELISA was done as per the user guide using Milliplex MAP Rat cytokine/chemokine magnetic bead panel kit (RECYTMAG_65K; Millipore; Temecula, CA, USA) and reading was done by LUMINEX MAGPIX® detection system with xPONET software (Millipore Corporation; Billerica, MA, USA). Sensitivity of TNF-α ranges between 1.9 to 156.3 pg/mL, of IL-1β ranges between 2.8 to 2,500.0 pg/mL and of IL-6 ranges between 30.7 to 4,687.5 pg/mL. For detecting the levels of hCDNF and rCDNF, Sandwich enzyme immunoassay was done using separate kits (SEG458Hu and SEG458Ra; Uscn Life Science Inc; Wuhan, Hubei, China) and absorbance was read at 450 nm using a Multiskan® MULTISOFT spectrophotometer (Labsystems; Tokyo, Japan).